Nitroxides for Use in Treating or Preventing Diabetes

ABSTRACT

Pharmaceutical compositions are provided that are useful in treating diabetes. The compositions comprise a pharmaceutically acceptable carrier, and an effective therapeutic or prophylactic amount of a nitroxide antioxidant that alters the expression of genes related to diabetes. Methods are also provided for the use of the pharmaceutical compositions in the treatment or prevention of diabetes. In a preferred embodiment, the nitroxide antioxidant is Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to pharmaceutical compositions useful fortreating or preventing diabetes, and to methods for using thesecompositions in treating or preventing diabetes.

2. Description of the Related Art

Diabetes mellitus is an umbrella term for a number of metabolicdisorders, all of which share the common symptom of hyperglycemia. It isclassified into two broad categories: type I, which generally resultsfrom autoimmune destruction of the pancreatic beta cells that produceinsulin and is characterized by insulin deficiency and a tendency todevelop ketosis, and type II, which results from a variety of geneticand metabolic defects in insulin action and/or secretion and ischaracterized by variable degrees of insulin resistance, impairedinsulin secretion, and increased glucose production.

Diabetes is prevalent worldwide and the incidence of the disease hasrisen dramatically over the past two decades. In the United States,approximately 6% of the population has diabetes, and approximately800,000 new cases occur each year. The prevalence of the disease isapproximately two times higher among African Americans, NativeAmericans, and Hispanic Americans that among non-Hispanic whites.

Individuals with diabetes are often unaware that they have the disorder;studies have suggested that type 2 diabetes may be present for up to adecade before diagnosis, and as many as half of type 2 diabetes patientshave one or more diabetes-related complications (e.g., eye disease,sensory and motor neuropathy, nephropathy, vascular disease,gastrointestinal or genitourinary dysfunction). Screening using afasting plasma glucose test is commonly employed to diagnose such“silent” cases of the disease.

Long-term management of diabetes is complex and the active participationof the patient is crucial for success. It involves individualizednutritional and exercise regiments, with patient self-monitoring ofglucose levels and, where appropriate, self-administration of insulinpreparations. Failure to comply with the therapeutic program is afrequent problem, and depression and eating disorders are oftenencountered.

Diabetes is, thus, a very common disease presenting serious potentialcomplications, which is difficult to manage. It would be desirable toavoid these issues by developing methods of directly curing theinsulin-related problems that give rise to them. One potential way to doso is to use genetic therapy to target the cellular dysfunction thatcauses diabetes. To this end, it would be desirable to identify genesrelated to diabetes and develop methods of altering the expressionpatterns of those genes so as to prevent the development of the diseaseor reduce its effects once it has occurred.

SUMMARY OF THE INVENTION

Pharmaceutical compositions are provided that are useful in preventingand treating diabetes. The compositions comprise a pharmaceuticallyacceptable carrier, and an effective therapeutic or prophylactic amountof an agent that changes the expression pattern of a gene related todiabetes. Methods are also provided for the use of the pharmaceuticalcompositions in the alteration of intracellular levels ofdiabetes-related proteins. In a preferred embodiment, the agent is anitroxide antioxidant, such as Tempol(4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

As described above, a composition and method are disclosed which areuseful in treating or preventing diabetes. In a preferred embodiment,the agent used to change the expression pattern of a gene related todiabetes is a nitroxide antioxidant. Tempol is a stable nitroxideradical characterized by the chemical formula4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl that has antioxidativeproperties. The present applicants have discovered that Tempol alsopossesses the novel property of altering the expression of genesencoding for proteins associated with the amelioration of diabetes (seeTables 1 and 2 below). Previous therapies have generally not focused onaltering the expression patterns of such diabetes-related genes.

The use of other nitroxide compounds is also contemplated. According tocertain embodiments the nitroxide compound can be selected from thefollowing formulas:

Wherein X is selected from O— and OH, and R is selected from COOH, CONH,CN, and CH₂NH₂.

Wherein X is selected from O— and OH, and R₁ is selected from CH₃ andspirocyclohexyl, and R₂ is selected from C₂H₅ and spirocyclohexyl.

Wherein X is selected from O— and OH and R is selected from CONH.

Wherein X is selected from O— and OH and R is selected from H, OH, andNH₂.

Suitable nitroxide compounds can also be found in Proctor, U.S. Pat. No.5,352,442, and Mitchell et al., U.S. Pat. No. 5,462,946, both of whichare hereby incorporated by reference in their entireties.

A non-limiting list of nitroxide compounds include:2-ethyl-2,5,5-trimethyl-3-oxazolidine-1-oxyl (OXANO),2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO),4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL),4-amino-2,2,6,6-tetramethyl-1-piperidinyloxy (Tempamine),3-Aminomethyl-PROXYL, 3-Cyano-PROXYL, 3-Carbamoyl-PROXYL,3-Carboxy-PROXYL, and 4-Oxo-TEMPO. TEMPO can also be substituted,typically in the 4 position, for example, 4-amino, 4-(2-bromoacetamido),4-(ethoxyfluorophosphonyloxy), 4-hydroxy, 4-(2-iodoacetamido),4-isothiocyanato, 4-maleimido, 4-(4-nitrobenzoyloxyl), 4-phosphonooxy,and the like.

Experimental Protocol

To assess the effects of Tempol on gene expression, Tempol wasadministered to experimental mice at a dose of 5 mg/g of food from 14months to 31 months after birth. Mice receiving the same food withoutthe addition of Tempol were used as a negative control. At the age of 31months, the experimental animals were sacrificed and the hearts weresurgically removed. The expression of a broad spectrum of genes in thecardiac tissue was assessed using chip-based microarray technology. Suchchips are well known in the art and are widely used to assess geneexpression. The experimental results showed that a gene related todiabetes, keratinocyte growth factor (KGF; also known as fibroblastgrowth factor-7), exhibited a more than twofold increase in expression.This gene is shown in Table 1.

In a further gene expression study, Tempol was administered toexperimental mice at a dose of 5 g/kg of diet from 12 months through 15months. Mice receiving the same diet without the addition of Tempol wereused as a negative control. At the age of 15 months, the adipose tissueof the experimental animals was obtained. The expression of a broadspectrum of genes in the adipose tissue was assessed using chip-basedmicroarray technology. Specifically, in this case an Affymetrix MOE430A2.0 array, containing 12,960 genes, was employed. Such chips are wellknown in the art and are widely used to assess gene expression. Theexperimental results on the adipose tissue show that a gene related todiabetes, adiponectin (C1Q and collagen domain containing) (ADIPOQ)exhibited an increase in expression. This gene is shown in Table 2.

TABLE 1 DIABETES-RELATED GENE EXIBITING INCREASED EXPRESSION IN CARDIACTISSUE AFTER TEMPOL ADMINISTRATION De- TEMPOL-treated scrip- Controlmice mice Fold ORF tion tpc1 tpc2 tpc3 tp51 tp52 tp53 change Z22703Kerati- 49 21 45 13 52 96 2.1 nocyte Growth Factor (Fibro- blast GrowthFactor 7)

TABLE 2 DIABETES-RELATED GENE EXHIBITING INCREASED EXPRESSION IN ADIPOSETISSUE AFTER TEMPOL ADMINISTRATION Mean (Tempol- Mean (Control FoldDescription treated mice) mice) P Value change Adiponectin, C1Q 3387627698 0.003 1.22 and collagen domain containing

A short summary of the genes described in Tables 1 and 2 is providedbelow.

Keratinocyte Growth Factor (Fibroblast Growth Factor 7)

KGF is a member of the heparin-binding fibroblast growth factor familyand was originally isolated from a human lung fibroblast cell line. Itexhibits a high degree of specificity for epithelial cells both in vitroand in vivo. A recent study investigated the effects of KGF on beta-cellgrowth and differentiation on islet-like cell clusters derived fromhuman fetal pancreas. (Movassat et al., Diabetologia 46:822-829 (2003))Although the exposure of human fetal beta-islet cells to KGF underculture conditions had no effect on the number of insulin producingcells as measured by insulin or DNA content, increased numbers of betacells were found in 8-week xenografts from Rowlett athymic nude ratsgiven KGF for 10 consecutive days beginning 48 hours after human fetalislet cell transplantation. This was found to be the result ofKGF-induced proliferation of pancreatic ductal cells and thedifferentiation thereof into functional beta cells. Moreover, whenKGF-treated animals were challenged with glucose, it was shown that thetransplanted cells were fully capable of producing insulin, unlike thecontrol group, in which the grafts were incompetent in this regard.Increasing the concentration of KGF, for example by the administrationof Tempol, thus offers the possibility of counteracting the pathologygiving rise to diabetes.

As shown in Table 1, the expression of HGF in the cardiac tissue of theexperimental mice was increased 2.1-fold in the animals treated withTempol.

Adiponectin, C1Q and Collagen Domain Containing

Adiponectin is an adipokine that is secreted specifically fromdifferentiated adipocytes. Adiponectin is found in serum atconcentrations up to 10 μg/ml. Adiponectin is present at reduced levelsin the plasma of patients with diabetes. Furthermore, administration ofexogenous adiponectin has been shown to correct metabolic defects thatare associated with insulin resistance. (Hug et al., Current Opinion inPharmacology 5 (2005) 129-134) A decrease in adiponectin level has beenimplicated in the development of insulin resistance. (Hotta et al.,Diabetes 50 (2001) 1126-1133) It has been suggested that adiponectin andTNF-α act in counter regulatory pathways and that the net balance oftheir actions results in the proper homeostasis of glucose and fattyacid metabolisms. When adiponectin levels decline in the serum, as forexample in obesity, the adipose TNF-α activity could lead to a shifttoward insulin resistance. (Hug et al., Current Opinion In Pharmacology5 (2005) at 131-132) Increasing the serum concentration of adiponectin,for example by the administration of Tempol, thus offers the possibilityof reestablishing the balance in the regulatory pathways and halting theshift toward insulin resistance.

As shown in Table 2, the expression of adiponectin in the adipose tissueof the experimental mice was increased 1.22-fold in the animals treatedwith Tempol.

Preferred Embodiment: Diabetes Prophylaxis and Treatment Protocol

As described above, Tempol has the effect of increasing the expressionlevel of KGF and adiponectin, genes related to diabetes. Since theexpression of these genes is increased, administration of Tempol willhave a beneficial effect by increasing concentrations of gene productsthat are beneficial in counteracting the disease. In a preferredembodiment of the present invention, therefore, Tempol is administeredto a mammalian host, such as a human, exhibiting no symptoms of diabetesin order to prevent the development of diabetes. Particularly preferredpatients are those who are predisposed or otherwise at risk fordiabetes, such as those with a family history of diabetes or those withgenetic or serum markers associated with diabetes. Alternatively, Tempolmay be administered to a human exhibiting diabetes in order toameliorate the effects of the disease on the patient. For this purpose,Tempol, non-toxic salts thereof, acid addition salts thereof or hydratesthereof may be administered systemically or locally, usually by oral orparenteral administration.

The doses to be administered are determined depending upon, for example,age, body weight, symptom, the desired therapeutic effect, the route ofadministration, and the duration of the treatment. In the human adult,the dose per person at a time is generally from about 0.01 to about 1000mg, by oral administration, up to several times per day. Specificexamples of particular amounts contemplated via oral administrationinclude about 0.02, 0.03, 0.04, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30,0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90,0.95, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135,140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205,210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275,280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345,350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415,420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485,490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555,560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625,630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695,700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765,770, 775, 780, 785, 790, 795, 800, 805, 810, 820, 825, 830, 835, 840,845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910,915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980,985, 990, 995, 1000 or more mg. The dose per person at a time isgenerally from about 0.01 to about 300 mg/kg via parenteraladministration (preferably intravenous administration), from one to fouror more times per day. Specific examples of particular amountscontemplated include about 0.02, 0.03, 0.04, 0.05, 0.10, 0.15, 0.20,0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80,0.85, 0.90, 0.95, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120,125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190,195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260,265, 270, 275, 280, 285, 290, 295, 300 or more mg/kg. Continuousintravenous administration is also contemplated for from 1 to 24 hoursper day to achieve a target concentration from about 0.01 mg/L to about100 mg/L. Specific examples of particular amounts contemplated via thisroute include about 0.02, 0.03, 0.04, 0.05, 0.10, 0.15, 0.20, 0.25,0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85,0.90, 0.95, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more mg/L. The dose to beused does, however, depend upon various conditions, and there may becases wherein doses lower than or greater than the ranges specifiedabove are used.

Tempol may be administered in the form of, for example, solidcompositions, liquid compositions or other compositions for oraladministration, injections, liniments or suppositories for parenteraladministration.

Solid compositions for oral administration include compressed tablets,pills, capsules, dispersible powders and granules. Capsules include hardcapsules and soft capsules. In such solid compositions, Tempol may beadmixed with an excipient (e.g. lactose, mannitol, glucose,microcrystalline cellulose, starch), combining agents (hydroxypropylcellulose, polyvinyl pyrrolidone or magnesium metasilicate aluminate),disintegrating agents (e.g. cellulose calcium glycolate), lubricatingagents (e.g. magnesium stearate), stabilizing agents, agents to assistdissolution (e.g. glutamic acid or aspartic acid), or the like. Theagents may, if desired, be coated with coating agents (e.g. sugar,gelatin, hydroxypropyl cellulose or hydroxypropylmethyl cellulosephthalate), or be coated with two or more films. Further, coating mayinclude containment within capsules of absorbable materials such asgelatin.

Liquid compositions for oral administration include pharmaceuticallyacceptable solutions, suspensions, emulsions, syrups and elixirs. Insuch compositions, Tempol is dissolved, suspended or emulsified in acommonly used diluent (e.g. purified water, ethanol or mixture thereof).Furthermore, such liquid compositions may also comprise wetting agentsor suspending agents, emulsifying agents, sweetening agents, flavoringagents, perfuming agents, preserving agents, buffer agents, or the like.

Injections for parenteral administration include solutions, suspensions,emulsions and solids which are dissolved or suspended. In injections,Tempol may be dissolved, suspended and emulsified in a solvent. Thesolvents are, for example, distilled water for injection, physiologicalsalt solution, vegetable oil, propylene glycol, polyethylene glycol,alcohol such as ethanol, or a mixture thereof. Moreover the injectionsmay also include stabilizing agents, agents to assist dissolution (e.g.glutamic acid, aspartic acid or POLYSORBATE80 (registered trade mark)),suspending agents, emulsifying agents, soothing agents, buffer agents,preserving agents, etc. They are sterilized in the final process ormanufactured and prepared by sterile procedure. They may also bemanufactured in the form of sterile solid compositions, such as afreeze-dried composition, and they may be sterilized or dissolvedimmediately before use in sterile distilled water for injection or someother solvent.

Other compositions for parenteral administration include liquids forexternal use, and ointment, endermic liniments, inhale, spray,suppositories for rectal administration and pessaries for vaginaladministration which comprise Tempol and are administered by methodsknown in the art.

Spray compositions may comprise additional substances other thandiluents: e.g. stabilizing agents (e.g. sodium sulfite hydride),isotonic buffers (e.g. sodium chloride, sodium citrate or citric acid).For preparation of such spray compositions, for example, the methoddescribed in U.S. Pat. No. 2,868,691 or No. 3,095,355 may be used.Briefly, a small aerosol particle size useful for effective distributionof the medicament may be obtained by employing self-propellingcompositions containing the drugs in micronized form dispersed in apropellant composition. Effective dispersion of the finely divided drugparticles may be accomplished with the use of very small quantities of asuspending agent, present as a coating on the micronized drug particles.Evaporation of the propellant from the aerosol particles after sprayingfrom the aerosol container leaves finely divided drug particles coatedwith a fine film of the suspending agent. In the micronized form, theaverage particle size is less than about 5 microns. The propellantcomposition may employ, as the suspending agent, a fatty alcohol such asoleyl alcohol. The minimum quantity of suspending agent is approximately0.1 to 0.2 percent by weight of the total composition. The amount ofsuspending agent is preferably less than about 4 percent by weight ofthe total composition to maintain an upper particle size limit of lessthan 10 microns, and preferably 5 microns. Propellants that may beemployed include hydrofluoroalkane propellants and chlorofluorocarbonpropellants. Dry powder inhalation may also be employed.

EXAMPLE 1

A 70-kilogram diabetic patient is administered a dose of 1500 mg ofTempol per day for 180 days. This may be administered in a single dose,or may be administered as a number of smaller doses over a 24-hourperiod: for example, three 500-mg doses at eight-hour intervals.Following treatment, the protein level of keratinocyte growth factor inthe pancreatic ductal cells, and the serum level of adiponectin, isincreased.

EXAMPLE 2

A 70-kilogram patient at risk for but not yet diagnosed with diabetes isadministered a dose of 1500 mg of Tempol per day for 180 days. This maybe administered in a single dose, or may be administered as a number ofsmaller doses over a 24-hour period: for example, three 500-mg doses ateight-hour intervals. Following treatment, the protein level ofkeratinocyte growth factor in the pancreatic ductal cells, and the serumlevel of adiponectin, is increased.

1. A method for increasing intracellular levels of one or more proteinsassociated with diabetes, comprising: identifying an individual in needof increasing levels of diabetes-associated proteins; and administeringto that individual an effective amount of a nitroxide antioxidant. 2.The method of claim 1, wherein the nitroxide antioxidant is4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl.
 3. The method of claim1, wherein the diabetes-associated protein is keratinocyte growthfactor.
 4. The method of claim 1, wherein the diabetes-associatedprotein is adiponectin.
 5. The method of claim 1, wherein the effectiveamount of a nitroxide antioxidant is within a range of 0.01-300 mg/kg.6. The method of claim 1, wherein the effective amount of a nitroxideantioxidant is within a range of 0.1-250 mg/kg.
 7. The method of claim1, wherein the effective amount of a nitroxide antioxidant is within arange of 1-200 mg/kg.
 8. The method of claim 1, wherein the effectiveamount of a nitroxide antioxidant is within a range of 2-150 mg/kg. 9.The method of claim 1, wherein the effective amount of a nitroxideantioxidant is within a range of 5-125 mg/kg.
 10. The method of claim 1,wherein the effective amount of a nitroxide antioxidant is within arange of 7-100 mg/kg.
 11. The method of claim 1, wherein the effectiveamount of a nitroxide antioxidant is within a range of 10-75 mg/kg. 12.The method of claim 1, wherein the effective amount of a nitroxideantioxidant is within a range of 15-30 mg/kg.
 13. A method forinhibiting the progression of diabetes associated with a protein,comprising: identifying an individual affected by or at risk for theprotein-associated diabetes; and administering to that individual anamount of a nitroxide antioxidant effective to increase expression of agene associated with the protein-associated diabetes.
 14. The method ofclaim 13, wherein the gene is keratinocyte growth factor.
 15. The methodof claim 13, wherein the gene is adiponectin.
 16. The method of claim13, wherein the nitroxide antioxidant is4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl.
 17. The method of claim13, wherein the effective amount of the nitroxide antioxidant is withina range of 0.01-300 mg/kg.
 18. The method of claim 13, wherein theeffective amount of the nitroxide antioxidant is within a range of0.1-250 mg/kg.
 19. The method of claim 13, wherein the effective amountof the nitroxide antioxidant is within a range of 1-200 mg/kg.
 20. Themethod of claim 13, wherein the effective amount of the nitroxideantioxidant is within a range of 2-150 mg/kg.
 21. The method of claim13, wherein the effective amount of the nitroxide antioxidant is withina range of 5-125 mg/kg.
 22. The method of claim 13, wherein theeffective amount of the nitroxide antioxidant is within a range of 7-100mg/kg.
 23. The method of claim 13, wherein the effective amount of thenitroxide antioxidant is within a range of 10-75 mg/kg.
 24. The methodof claim 13, wherein the effective amount of the nitroxide antioxidantis within a range of 15-30 mg/kg.
 25. A method for treating diabetes,comprising: administering to a diabetes patient an amount of a nitroxideantioxidant effective to increase proliferation of pancreatic beta cellsor ameliorate insulin resistance.
 26. The method of claim 25, whereinthe nitroxide antioxidant is administered in an amount effective toincrease intracellular levels of at least one protein related toproliferation of pancreatic beta cells.
 27. The method of claim 25,wherein the nitroxide antioxidant is administered in an amount effectiveto increase intracellular levels of at least one protein related to theamelioration of insulin resistance.
 28. The method of claim 25, whereinthe effective amount of the nitroxide antioxidant is within a range of0.01-300 mg/kg.
 29. The method of claim 25, wherein the effective amountof the nitroxide antioxidant is within a range of 0.1-250 mg/kg.
 30. Themethod of claim 25, wherein the effective amount of the nitroxideantioxidant is within a range of 1-200 mg/kg.
 31. The method of claim25, wherein the effective amount of the nitroxide antioxidant is withina range of 2-150 mg/kg.
 32. The method of claim 25, wherein theeffective amount of the nitroxide antioxidant is within a range of 5-125mg/kg.
 33. The method of claim 25, wherein the effective amount of thenitroxide antioxidant is within a range of 10-75 mg/kg.
 34. The methodof claim 25, wherein the effective amount of the nitroxide antioxidantis within a range of 15-30 mg/kg.
 35. The method of claim 26, whereinthe protein is keratinocyte growth factor.
 36. The method of claim 27,wherein the protein is adiponectin.
 37. Use of a nitroxide antioxidantin the preparation of a medicament for increasing intracellular levelsof one or more proteins associated with diabetes.
 38. Use of a nitroxideantioxidant in the preparation of a medicament for inhibiting theprogression of diabetes associated with a protein.
 39. Use of anitroxide antioxidant in the preparation of a medicament for treatingdiabetes.
 40. Use of a nitroxide antioxidant in the preparation of amedicament for increasing proliferation of pancreatic beta cells orameliorating insulin resistance.